The Research Of Induced Differentiation Of Bone Marrow Stromal Cells And Improving Functional Outcome Following Bone Marrow Stromal Cells Transplantation In Injured Spinal Cord In Rats

ObjectiveInvestigate the feasibility of in vitro - induced and vivo - induced differentiation of bone marrow stromal cells ( BMSCs) into neural cells and to observe the affection on neurological functional recovery of transplantation of BMSCs in injured spinal cord in adult rats.MATERIAL AND METHODANIMAL: Forty - one healthy adult wistar rats, weighing 250 to 350g each , were used for the experiments. Female and male rats all can be concluded. The rats are randomly divided into four groups: ( 1) 14 rats, left spinal cord he-misection and transplantation of BMSCs after 7 days; (2) 14 rats,left spinal cord hemisection with injection of phosphate buffered saline after 7 days; (3)10 rats, left spinal cord hemisection alone; (4) 3 rats, sham - operation. Four young wistar rats, weighing 100g each, were used for the cultivation of BMSCs.MATERIALS: L-DMEM(Gibico) , all - trans - retinoic acid(RA) ,brain derived neural factor ( BDNF) ( SIGMA ) ; primary antibodies against Brdu, NSE, GFAP (rabbit polyclonal) (Boster,wuhan) ,et al.EQUIPMENT: CO₂ incubator, superclean bench, inverted microscope and light microscope, micrography ( OLYMPUS, JAPAN ) , stereotaxic apparatus, six - bore culture board, 25ml culture flask, cells counting plate.METHODS:1. Four young wistar rats were killed by broken - neck. BMSCs were collected from femurs by flushing the shaft with DMEM ( with 10% fetal bovine serum). Finally the BMSCs were cultivated in the 25 ml culture flask with a concentration of 1 x 109 cells/L. The cells were cultivated in the 37 ,5% CO2 incubator. When the cells proliferated to the extent of 90% to make subculturing.2. BMSCs were cultivated in a medium containing RA and BDNF as induction medium. The morphological changes of the cells were observed. At 14 day of induction, the cells were stained immunocytochemically with GFAP and NSE antibodies.3. After 7 days BMSCs and PBS were injected to injured spinal cord in adult rats. Functional outcome measurements were performed using BBB score at 24 hours before transplantation and 1,7,14,21, 28 days after transplantation, and to observe the recovery of nerve function in rats.4. Spinal cord tissues were prepared for HE staining to detect the chang of histology, and for immunohistochemical and immunofluorescent staining to detect whether the Brdu - labeled BMSCs expressed the astrocytic marker - GFAP( gli-al fibriliary acidic protein) or the neuron marker - NSE (neuron - specific eno-lase).RESULT1. Original cultivation and subculturing of BMSCs: when original cells were cultured, cluster - like clones appeared, and maldistribution . After subculturing , BMSCs proliferated quickly. BMSCs were almost full of the culture flask 3-4days later, and the cells were spindle in shape and well - distributed.2. At 24 hours of induction by RA and BDNF, The morphous of BMSCs were obviously different . The postinduction BMSCs were extremely similar to the neural cells. The cells were stained immunocytochemically with GFAP and NSE antibodies after induction 14 days. Most cells were positive in NSE and GFAP and was brown, the positive rate of NSE was 79.7% ±2.6% ; the positive rate of GFAP is 66. 8% ±5. 7%. The cells of control group expressingNSE,GFAP was very few.3. Evaluation of Ethology: 1 — 4weeks after operation, the nerve function of all animals recoveryed with differential extent, but the animals of PBS group acted slowly, and response was slow, and aconuresis or retention of urine was obvious, Mortality was 42. 6%. Only one animal of the BMSCs group died. The mortality of the two groups is obviously differential. Five animals of the spinal cord hemisection alone group died. The mortality is 50% , and compared with PBS group had no significant difference ( p > 0.05 ).Functional outcome measurements were performed using BBB score at 24 hours before transplantation and 1,7,14, 21, 28 days after transplantation . At 24 hours before transplantation and 1,7 days the score of BMSCs and PBS group had no significant difference (p >0. 05). At 14 days the difference of the two groups was very obvious (p <0. 05). The spinal cord hemisection alone group and PBS group had no significant difference ( p > 0. 05 ). At 28 days BMSCs group and sham - operation group had significant difference( p < 0.05 ).4. Histology of transplantation: the result of HE staining: at 1 days of transplantation, few cells became degeneration and necrosis. At 7days, necrotic tissue became small capsular space . The transplanted cells was small and anach-romasis, and filled in the injuried space. At 14 days, the transplanted cells became large, and some cells had apophysis. At 28 days, the transplanted cells were fused with gray nucleus. The untransplanted slice can be seen that some tissue became spareness , necrosis , liquefaction and injuried space.The immunocytochemical result : BMSCs that cell nucleus was brown survived in the spinal cord of BMSCs group. At 7,14,21,28 days, the average cells amounts was 117.2 ±38.14,94.50 ±33.34,82.15 ±26.40 ft 69.5 ±22. 76. By immune double - stained methods, some of the BrdU - labeled cells started to express NSE and GFAP at 7 days of transplantation . Soon after the percentage would rise, it was 9.1% ft 2.4% at 28 days.CONCLUTIONBMSCs can be induced and differentiated into neural cells. Transplantation...